1. Field of the Invention
The invention relates to compositions comprising membrane-derived microparticles that promote platelet adhesion, aggregation, blood clot formation and to a method for treating bleeding disorders, particularly blood coagulation disorders, platelet function disorders in the instance of low platelet counts. The invention is also useful in minimizing blood loss in a mammal, in particular in patients with trauma, patients undergoing surgical or medical invasive procedures where blood loss can be substantial.
2. Background
(A) Clinical Bleeding Disorders
(B) Cell-Derived Microparticles (C-MP).
2(A). Clinical Bleeding Disorders
A number of medical disorders manifesting prolonged bleeding can be classified into (i) platelet disorders, (ii) clotting disorders and (iii) ill defined bleeding disorders.
(i) Platelet Disorders. Patients with platelet disorders often bleed excessively because number of platelets are insufficient (thrombocytopenia) or platelet function is impaired, although platelet counts are normal (platelet dysfunction). For example, patients with idiopathic thrombocytopenic purpura (ITP) have insufficient platelets and suffer from easy bruising and bleeding. Thrombocytopenia (low platelet counts) may be caused by medications, especially following intensive chemotherapy regimens. Many drugs, e.g. aspirin, impair platelet function, leading to excessive bleeding although patients platelet counts may be normal. Some systemic illness also may cause acquired form of platelet disorders. Thrombocytopenias or platelet dysfunctions may be congenital or acquired.
(ii) Clotting Disorders. A group of genetic bleeding disorders, including von Willebrand disease (vWd) and other coagulation disorders, are characterized by the commonality of prolonged bleeding. In many of these patients with clotting disorders there is an insufficiency in the amount and/or function of clotting factors such as factor eight (FVIII) or von Willebrand factor (vWf) in blood or other clotting factors. vWf promotes platelet adhesion, aggregation and carries clotting factor VIII (FVIII). FVIII is essential for generation of thrombin and subsequent blood coagulation.
(iii) There are many ill defined bleeding disorders, some of which involve blood vessel wall.
Although various treatments are available for bleeding disorders, there is a need for treatment methods with increased efficacy and/or reduced side effects.
For example, currently, transfusion of allogeneic blood products (blood products from donors, not the patient) such as platelets and coagulation factors is the mainstay of treatment of bleeding disorders. However, such agents pose a risk of transmitting blood borne infections such as hepatitis, HIV virus and can induce immune mediated transfusion reactions. To circumvent these problems, production of recombinant clotting factors or effective small molecules is actively pursued. Some such agents are available for clinical use (Coagulin-A®, Kogenate®, for example), however, these are still very expensive and all patients are not responsive to these agents. Other treatments comprise administering human purified vWf or a cryoprecipitate of human plasma containing vWf. Directly injected vWf has a very short clearance time, further complicating therapy. Moreover, some patients reject foreign proteins and do not respond to these replacement therapies. Other hemostatic agents such as desmopressin (DDAVP) are used to treat some patients of hemophilia and vWd, but many patients do not respond to this treatment. Thus there remains a need for new treatments for bleeding disorders wherein spontaneous or trauma induced bleeding are frequent complications, sometimes life threatening.
The present invention addresses all of these problems by development of an effective, safe treatment using autologous blood products or heterologous blood products with reduced capacity for transmission of blood borne pathogen and reduced capacity of immune response induction. This method will improve the quality of life in patients with bleeding disorders and save many lives and additionally resolve a serious shortage of blood supply, an increasing problem in transfusion medicine.
2(B). Cell-derived Microparticles (MP)
Release of cell membrane-derived microparticles (MP) has been demonstrated during cell activation or apoptosis. Release of MP has been demonstrated from platelets (PMP), leukocytes (LMP), red cells (RMP) and endothelial cells (EMP) (1-3). Most MP expose procoagulant anionic phospholipids such as phosphatidylserine (PS) (4-7), having platelet factor 3 activity (PF3) (8). This activity is believed to be a major function of MP in vivo. More recently, tissue factor (TF) has been identified on leukocyte MP (LMP) (9-11), endothelial MP (EMP) (11-14), and platelet MP (PMP) (10, 15) further suggesting important roles in hemostasis and thrombosis. Prior this disclosure, TF has not been localized on RMP.
The current inventors have previously demonstrated that PMP, LMP, EMP play important roles in hemostasis and thrombosis and inflammation (1-3). They reported that surface antigens on EMP are distinctive but vary depending on the type of endothelial cell injury, e.g. apoptosis, activation (34). EMP have been shown to exhibit procoagulant activity and to be related to many thrombotic and inflammatory conditions, including lupus, MS and other chronic inflammatory disorders (2, 3, 10). EMP is further known to carry tissue factor (TF). Nevertheless, a functional role of MP in disease amelioration has not been disclosed. It was the hypothesis of the current inventors that because MPs are procoagulant, they could be exploited as therapeutic agents in bleeding disorders (3).
Coller and colleagues conjugated RGD to red cells to make them hemostatically active and named the product thromboerythrocytes in 1992 [(53) and U.S. Pat. No. 5,328,840)]. However, the Coller invention does not disclose nor anticipate the uses of the microparticles of the present invention. Other hemostatic liposome preparations have been reported, including conjugation of clotting factors such as FVIII (54,55) and fibrinogen (56,57) to them or albumin particles to produce hemostatically active vesicles.
Chemical modifications of artificial liposomes include platelet specific adhesion molecules such as GpIIb/IIIa and Ib/IX conjugated to synthetic liposomes or to albumin to produce “artificial platelets” (58-61).
There are disadvantages to these approaches. Synthetic microparticles may activate complement cascade, creating adverse complications and may induce immune reactions, leading to autoimmune responses. No method using synthetic MP or blood cells has yet emerged as clinically practicable and there are little follow-up clinical studies.
The present invention employs novel microparticles (MP), in particular red cell microparticles (RMP), as agents for this purpose. In one embodiment, cell derived microparticles are conjugates to a protein or factor absent in a patient. In the preferred embodiment, autologous cell-derived red cell microparticles are used as hemostatic agents. This embodiment of the method has clear advantages: because the composition is autologous, one avoids immune system activation often cause by allogeneic transfusions or synthetic substances and eliminates risk of transmission of blood-borne pathogenic agents. Because red cells are the most abundant blood cell, a small fraction of blood (50-100 ml of blood from whole blood volume over 5000 ml) can be drawn safely from patients and RMP can be generated to be infused back to the same patients. The supply of the composition is safe and convenient for the patient.